Name: 4d microscopy of yeast
Uploaded: 2019-04-28T13:00:00
Description: Watch this Scientific Journal Video about 4D Microscopy of Yeast at JoVE.com

这项工作得到了NIH赠款R01 GM104010的支持。感谢在综合显微镜核心设施为Vytas Bindokas和Christine Labno提供荧光显微镜帮助,该设施由NIH资助的癌症中心支持赠款P30 CA014599提供支持。

1. 准备
<ol><li>使非荧光最小 NSD 介质2.核黄素的缺乏有望减少细胞内背景绿色荧光和相关光毒性。</li>
	<li>在+23°C下一夜之间将酵母菌株生长在5mL NSD中的对数相中,在50 mL的挡争议烧瓶中具有良好的曝气。分析前约3-4小时,稀释新鲜NSD中的酵母培养,使最终的OD600在成像时为0.5-0.8。</li>
	<li>制备2mg/mL的康卡纳林A(ConA)溶液。如果需要,将此溶液的等分液冷冻在液氮中,并储存在-80°C。</li>
	<...

<ol>
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酵母细胞器的4D共聚焦成像需要仔细调整多个参数。主要问题是光漂白和光毒性。典型的 4D 影片涉及收集数千个光学部分,因此激光照明必须保持在尽可能低的位。串联荧光蛋白标记可用于增强信号,而不会增加标记蛋白16、17的表达。最大化扫描速度有助于限制光损伤,还允许在适当的短间隔内捕获 Z 堆栈。在XY和Z中,在奈奎斯特极限处的体素尺寸最小化了...

内膜系统的隔间在不断变化,其完整的表征需要活细胞成像。此处描述的是使用 4D(延时 3D)共聚焦显微镜来可视化萌芽酵母中荧光标记的隔间的协议。该方法被开发来跟踪皮基亚牧师和萨查莫西斯塞雷维西亚1,2,3的分泌隔间的动态。该协议侧重于S.cerevisiae,它有一个非堆叠的Golgi,其中单个蓄水池是光学上可解析的4。S.cerevisiae的不寻常的戈尔吉组织通过 4D 显微镜演示了 Golgi csterna 最初与居民早期戈尔吉蛋白贴标签,然后在获取居民晚期戈尔吉蛋白 3 时丢失这些蛋白质 , 5.这种过渡可以通过创建一个菌株来可视化,其中早期的Golgi蛋白Vrg4被标记为GFP,而晚期的Golgi蛋白Sec7则标有单体红色荧光蛋白。当跟踪单个蓄水池时,成熟被观察为绿色到红色转换3。这种类型的分析可以提供有关蛋白质定位和隔间标识的宝贵信息。例如,两种稍微偏移到达和离开时间的蛋白质有时在静态图像中可能显示标记不同的隔间,但在 4D 电影中可以看到,可以在不同时间点6、7 标记同一隔间.因此,4D 显微镜揭示了其他不明显的现象。
酵母舱的信息4D显微镜可以通过适当的程序和设备2实现。只要有可能,荧光蛋白标记通过基因替换8执行,以避免过度表达伪影。由于细胞内结构通常非常动态,因此需要 4D 成像,以确保随着时间的推移可靠地跟踪结构。此处描述的协议采用激光扫描共聚焦显微镜,配备高灵敏度探测器。使用此装置,S.cerevisae的整个细胞体积大约每 1-3 秒可通过共聚焦显微镜成像,通常为 2 秒间隔。根据荧光水光水线的标签密度及其光物理特性,可以收集长达 5-15 分钟的数据。主要障碍是尽量减少光漂白。为此,激光强度尽可能低,共聚焦扫描速度最大化,光学参数配置为在奈奎斯特极限下成像,以便捕获相关信息,同时避免过度的光线照射。这些设置也有望减轻光毒性,在活细胞成像9,10,11中经常被忽视的因素。由此产生的噪声数据使用漂白剂校正和去卷积算法进行处理,以促进荧光强度的量化。
即使使用高质量的 4D 电影,分析也十分棘手,因为酵母隔间往往数量众多、异构且可移动。由于共聚焦显微镜的内在局限性以及长时间 4D 成像所需的非最佳设置,彼此接近的荧光结构难以解析。这个问题可以通过关注在标签期间保持光学可解的少量荧光结构来规避,并假定这些结构代表标签的全体人群车厢。通过观看投影 Z 堆栈的影片和创建一系列蒙太奇,将每个时间点的光学部分排列在计算机屏幕上,可以可靠地跟踪的荧光隔间可以识别。此分析采用自定义 ImageJ12插件,允许单独跟踪单个结构。
最近的方法论文涵盖了荧光蛋白在酵母13中的使用,以及酵母细胞2的4D共聚焦成像的理论与实践。该协议侧重于 4D 成像实验的关键实际方面。它包括以前描述的过程的一些增强功能,以及 ImageJ 插件代码和文档的更新版本。所示示例侧重于 Golgi 动力学,但此协议同样适用于成像其他酵母腔。

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	<tbody>
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			<td height="42" style="height:42px;width:216px;">35 mm glass bottom dishes No. 1.5</td>
			<td style="width:85px;">MatTek</td>
			<td style="width:119px;">P35G-0.170-14-C</td>
			<td style="width:167px;">Imaging dishes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Concanavalin A powder</td>
			<td style="width:85px;">Sigma-Aldrich</td>
			<td style="width:119px;">C2010</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Trolox</td>
			<td style="width:85px;">Vector Laboratories</td>
			<td style="width:119px;">CB-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Leica SP8 confocal microscope</td>
			<td style="width:85px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Leica Application Suite X&#160;</td>
			<td style="width:85px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>Microscope software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Huygens Essential software, version 17.04</td>
			<td style="width:85px;">Scientific Volume Imaging</td>
			<td style="width:119px;"></td>
			<td>Deconvolution software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td style="width:85px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Image processing and analysis software</td>
		</tr>
	</tbody>
</table>

4d microscopy of yeast

该协议的目的是描述膜腔在萌芽酵母的活细胞中如何形成和转化。酵母中的许多细胞内腔是动态的,要充分了解其特性,需要延时成像。多色 4D 共聚焦荧光显微镜是一种强大的方法来跟踪细胞内隔间的行为和组成,时间尺度为 5-15 分钟。 严格分析隔间动力学需要捕获数千个光学部分。为了实现这一目标,光漂白和光毒性通过以极低的激光功率快速扫描而最小化,像素尺寸和 Z 步长间隔设置为与全分辨率采样图像兼容的最大值。生成的 4D 数据集是嘈杂的,但可以通过反卷积来平滑。即使使用高质量的数据,分析阶段也极具挑战性,因为细胞内结构通常数量众多、异构且具有流动性。为了满足这一需求,将自定义 ImageJ 插件写入计算机屏幕上的阵列 4D 数据、识别感兴趣的结构、编辑数据以隔离单个结构、量化荧光时间课程以及制作投影 Z 堆栈的影片。4D 电影特别适用于区分稳定隔间与通过成熟而翻过来的瞬态隔间。此类电影还可用于描述诸如隔间融合等事件,并测试特定突变或其他扰动的影响。

这里给出的例子文档并量化了双色4D共聚焦显微镜3所示的两个酵母Golgi蓄水池的成熟。酵母细胞含有10-15 Golgi蓄水池的顺序,每个细胞在大约2-4分钟的一段时间内成熟。 成熟可以通过用GFP标记早期的Golgi标记Vrg4和用红色荧光标记已故的Golgi标记Sec7来可视化蛋白质,如mCherry或mscarlet。单个蓄水池标签最初带有 Vrg4 标记,然后经历一个短暂的转换,其中标记被交换,...

Watch this Scientific Journal Video about 4D Microscopy of Yeast at JoVE.com

4D Microscopy of Yeast

酵母的4D显微镜

该协议描述了使用多色4D(延时3D)共聚焦显微镜对萌芽酵母中荧光标记细胞内腔的分析。选择成像参数是为了捕捉足够的信号,同时限制光损伤。自定义 ImageJ 插件允许跟踪和定量分析标记的结构。

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

这种方法在几分钟内从三维中跟踪酵母细胞的结构，使我们能够研究细胞内细胞器和隔间的动态。4D 成像可确保我们可以得出有关细胞内动力学的可靠结论，尤其是对于可能是瞬态的结构或标记。演示这个程序的将是娜塔莉·约翰逊，一个来自我实验室的博士后。首先在5毫升非荧光合成定义，或NSD，中等，在15毫升的困惑烧瓶中生长感兴趣的酵母菌株的隔夜培养，在23摄氏度。分析前三到四个小时，在新鲜的NSD介质中稀释对数相酵母培养，使600纳米或OD600的最终光学密度在成像时为0.5至0.8。在培养物准备好前至少一小时，将每毫升 Concanavalin A 溶液的等同物全速离机五分钟，以颗粒任何颗粒，并将 250 微升的上清液加入一个干净的 35 毫米玻璃底部显微镜盘中。15分钟后，用两毫升蒸馏水洗两到三次，在干后加入250微升酵母培养物。等待10分钟，让细胞粘附，然后轻轻地用两毫升的新鲜NSD介质轻轻洗盘子两到三次。然后用两毫升的新鲜 NSD 覆盖细胞。要对酵母细胞进行成像，请在共焦光显微镜上选择一个 63 倍油浸透镜，其数值孔径至少为 1.4。在这里，还可以使用 100 倍的客观镜头，而不是 63 倍的客观镜头。然后，将盘子放在客观镜上，用浸入油发现。从"采集模式"选项卡的下拉菜单中，选择 xyzt。在 XY 选项卡下，将帧大小格式化为 256 宽度 X128 高度。使用最大扫描速度，通常为 8 千赫。如果双向 X 扫描可用，请打开该扫描，并将缩放系数调整为 9，这将导致像素大小约为 80 纳米。如果使用 100 倍目标，请相应地调整缩放系数，以保持大约 80 纳米的像素大小。然后将线累积设置为 4 或 6。将针孔设置为 1.2 个 Airy 单元，并打开白光激光器（如果有）。然后设置每个荧光通道的激发波长和激光功率百分比。如果可用，则通过取消选择最大集成时间，在配置选项卡下启用光子计数模式。对于每个荧光通道，分配一个高灵敏度检测器，设置发射波长范围，并打开光子计数模式（如果可用）。将每个荧光通道的时间浇注窗口设置为 0.6 到 10 纳秒，以避免从玻璃盘捕获反射光。接下来，打开亮场成像，并选择低灵敏度检测器进行数据收集。打开实时成像模式，并在明亮的场通道中打开增益，直到细胞清晰可见。如果在光子计数模式下，将灰色值的范围从手动更改为自动以查看荧光信号。将 Z 堆栈设置为成像酵母细胞的整个体积，并指定成像的方向性，以便向下向盖滑移动。将 Z 步长间隔设置为 0.25 至 0.35 微米，以获取每个 Z 堆栈大约 20 到 25 个光学部分，并在可用时打开 Galvo 流。对于典型的影片，将 Z 堆栈之间的时间间隔设置为两秒钟，将影片持续时间设置为 5 到 10 分钟。然后将影片另存为 LIF 文件。对于电影去卷积，启动一个适当的去卷积软件程序，使用经典的最大可能性估计算法，并打开数据系列。选择"去卷积"向导和参数编辑器，以确认已检测到并正确显示成像参数。将嵌入介质折射率值更改为 1.4 以近似酵母细胞质。然后使用编辑器估计盖滑位置。选择"设置所有已验证"并单击"接受"，然后选择"输入向导"。单击下一个箭头可绕过点传播函数选择和裁剪预处理阶段，然后通过每个荧光通道的去卷积向导。选择计算对数垂直映射功能并检查原始数据荧光强度剖面。将手动设置为背景估计模式，输入背景值，然后单击"接受"。将最大迭代值保留为 40，并输入估计信号与噪声比。然后关闭漂白剂校正，然后单击"去旋转"。如果噪声已充分消除而不消除昏暗结构中的正荧光，请选择"接受到下一个通道"。一旦所有荧光通道都令人满意地去配置，单击"全部完成"，然后先排列红色通道，然后是绿色、蓝色和明亮的通道，以便随后在 ImageJ 中进行编辑。然后将图像序列保存为八位 TIF 文件，每个通道选择一个文件，将对比度拉伸作为转换模式。要进行漂白校正，请将去旋转的图像序列导入 ImageJ，然后单击"图像"、超堆栈和堆栈到超堆栈，将图像转换为超堆栈。从下拉菜单中选择 xyzct，并输入通道数、Z 堆栈切片和时间帧数。要校正照片漂白的荧光通道，请选择"图像、颜色、分割通道"和"荧光通道"，请选择插件、EMBL 工具、漂白校正和指数拟合。然后选择"图像、颜色"和"合并通道"，将亮场和漂白校正荧光通道合并到超堆栈中，并将去旋转和漂白校正的超堆栈保存为后续影片生成和编辑，作为八位 TIF 文件。要将去旋转和漂白校正数据集转换为缩放蒙太奇，请选择插件、IJ_Plugins 和"制作蒙太奇系列"，然后选择相关的八位超堆栈。单击"打开"和"确定"以接受所有切片将用于创建蒙太奇，然后再次单击"确定"以接受建议的规模因子值。将原始蒙太奇保存为八位 TIF 文件，并选择插件、IJ_Plugins、蒙太奇系列到超堆栈和 OK，以创建一个包含所有时间帧的原始蒙太奇的 4D 超堆栈。要生成原始平均投影影片，请首先选择"IJ_Plugins、项目超堆栈和确定"，以接受 Z 投影默认参数。将平均投影影片保存为八位 TIF 文件，并检查影片以识别可以在其标记期间跟踪的单个结构。识别可在影片持续时间内可靠跟踪的结构，并隔离该结构进行分析，是该过程最关键的步骤。然后按照插件用户指南中的说明，通过编辑蒙太奇来隔离感兴趣的结构。从编辑蒙太奇的期间（包括感兴趣的结构）创建 4D 超堆栈，并保存编辑的超堆栈。要量化编辑的 4D 超堆栈中隔离结构的荧光强度，请选择插件、IJ_Plugins 和"分析已编辑的影片"，输入 Z 堆栈之间的时间间隔，然后单击"确定"。若要创建隔离结构的最终影片，请选择"插件、IJ_Plugins"和"项目超堆栈"，指定应包含的 Z 堆栈切片，选择"平均强度"作为"投影"类型，然后单击"确定"。若要在编辑的数据上使用原始数据创建 4D 超堆栈，请选择插件、IJ_Plugins、合并两个超堆栈，并将第一个放在第二位以上。若要在编辑的数据上使用原始数据从 4D 超堆栈生成影片，请选择"插件、IJ_Plugins"和"项目超堆栈"，请指定应包含的 Z 堆栈切片，选择"平均强度"作为投影类型，然后单击"确定"。在这里，原始数据的 Z 堆栈投影的第一帧可以与相同的去卷和漂白校正数据进行比较。这些帧从相同的去相和漂白校正电影显示两个蓄水池进行分析，其中首先标记与绿色Vanadate耐糖蛋白4或Vrg4标记，后来与红色分泌7或Sec7基因传输蛋白标记。此蒙太奇由去卷和漂白校正的超堆栈创建，在编辑之前和之后的单一时间点显示所有光学部分，以便从所选的蓄水池之一隔离信号。在此图中，投影 Z 堆栈的最后影片中的几帧显示在图的顶部，并在底部编辑投影。从所选的 Golgi cisternae 对绿色和红色荧光信号进行量化后，绿色 Vrg4 标记到达并持续约 80 秒，之后，红色 Sec7 标记到达并持续约 60 秒，两个标记之间短暂重叠。执行此过程时，确定可以在其生存期内明确跟踪的结构非常重要。在发生突变或某种其他类型的特定扰动后，可以执行相同类型的分析。该技术为研究高尔吉素酸和内质性沉默性退出位点以酵母为模型系统的动态行为开辟了一条。

Research

JoVE Journal

Biology

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...